Nitric oxide (NO) and protein S-nitrosylation (SNO) have been shown to play important roles in ischemic preconditioning (IPC)-induced acute cardioprotection. The majority of proteins that show increased SNO following IPC are localized to the mitochondria, and our recent studies suggest that caveolae transduce acute NO/SNO cardioprotective signalings in IPC hearts. Due to the close association between subsarcolemmal mitochondria (SSM) and the sarcolemma/caveolae, we tested the hypothesis that SSM, rather than the interfibrillar mitochondria (IFM), are major targets for NO/SNO signaling derived from caveolae-associated eNOS. Following either control perfusion or IPC, SSM and IFM were isolated from Langendorff perfused mouse hearts, and SNO was analyzed using a modified biotin switch method with fluorescent maleimide fluors. In perfusion control hearts, the SNO content was higher in SSM compared to IFM (1.330.19, ratio of SNO content Perf-SSM vs Perf-IFM), and following IPC SNO content significantly increased preferentially in SSM, but not in IFM (1.720.17 and 1.070.04, ratio of SNO content IPC-SSM vs Perf-IFM and IPC-IFM vs Perf-IFM, respectively). Consistent with these findings, eNOS, caveolin-3 and connexin-43 were detected in SSM, but not in IFM, and IPC resulted in a further significant increase in eNOS/caveolin-3 levels in SSM. Interestingly, we did not observe an IPC-induced increase in SNO or eNOS/caveolin-3 in SSM isolated from caveolin-3-/- mouse hearts, which could not be protected with IPC. In conclusion, these results suggest that SSM are the major target for protein SNO in the IPC mouse heart, suggesting that the SSM may be the preferential target of sarcolemmal signaling-derived post-translational protein modification (caveolae-derived eNOS/NO/SNO), thus providing an important role in cardioprotection. Our previous study in mouse embryonic fibroblasts showed that cysteine 202 of cyclophilin D (CyPD) is necessary for redox stress-induced activation of the mitochondrial permeability transition pore (mPTP). To further investigate the essential function of this cysteine residue in situ, we used CRISPR to develop a knock-in mouse model (C57BL/6N stain), where CyPD cysteine 202 was mutated to a serine (C202S-KI). The amount of total CyPD expressed in the CyPD C202S-KI did not differ compared to the wild-type (WT). However, the CyPD C202S-KI mouse hearts elicit a significant cardioprotective effect against ischemia-reperfusion (I/R) injury in the Langendorff perfused heart model. After 20 min of global ischemia followed by 90 min of reperfusion, the post-ischemic recovery of rate pressure product (RPP= heart rate x LVDP) was 45.04.2% in CyPD WT and 59.64.0% in CyPD C202S-KI mice. Myocardial infarct size was decreased in CyPD C202S-KI mouse hearts versus CyPD WT mice (24.54.7% vs 49.82.7%). Isolated heart mitochondria from CyPD C202-KI mice had a higher calcium retention capacity compared to CyPD WT mice (140.020.82 vs 213.316.67 umol Ca+2/g protein). However, in contrast to CyPD knockout mice which exhibit more pronounced cardiac hypertrophy in response to pressure overload stimulation than control mice, CyPD C202S-KI mice developed a comparable level of hypertrophy to their WT littermate in an angiotensin II-induced hypertrophy model delivered by implanted osmotic minipumps. In conclusion, these results show that mutated CyPD C202S affords cardioprotection against I/R injury, suggesting that the redox-modification of cysteine 202 might play an important role in the regulation of CyPD and its downstream targets such as mPTP.